Influenza-complicated methicillin-resistant S. aureus (MRSA) infection has emerged as a leading cause of death during recent influenza pandemics and epidemics. Both heightened bacterial burden and exaggerated lung inflammation are believed to be responsible for high mortality in patients and animal models. However, a hitherto incomplete understanding of co-infection pathophysiology has slowed the development of effective treatment strategies. NADPH oxidase 2 (NOX2) is an enzyme complex predominantly expressed by phagocytes. NOX2 produces superoxide which reacts with nitric oxide and results in the formation of highly toxic peroxynitrite. Therefore, both NOX2 and inducible nitric oxide synthase (iNOS) activities critically contribute to oxidative stress which has long been known to be involved in inflammatory lung damage. On the other hand, reactive oxygen species (ROS) generated by NOX2 activity has been shown to exacerbate influenza virus-induced tissue inflammation but to contribute to resistance to S. aureus lung infection~ whereas iNOS activity plays a detrimental role during influenza infection, it is dispensable for pulmonary S. aureus clearance. Preliminary studies in the PI's laboratory revealed that influenza infection reduces phagocyte ROS level but increases IFN-?/iNOS expression. Therefore, it is hypothesized that dysregulation of oxidative burst following influenza infection causes defective bacterial killing s well as excessive oxidative stress, and results in fatal influenza and MRSA co-infection. The approaches to test the hypothesis include: 1) determine the influence of influenza infection on NOX2 activity and its contribution to influenza-suppressed phagocytic MRSA killing. Particularly, the molecular mechanisms for influenza-suppressed NOX2-dependent MRSA clearance will be examined in selected gene- deficient mice~ 2) determine the contribution of oxidative stress to lung injury during lethal influenza and MRSA co-infection. Specifically, the synergistic or overlapping contribution of NOX2 versus IFN-?/iNOS activity to oxidative tissue damage will be examined in NOX2, IFN-?R and iNOS gene-deficient mice~ 3) refine combination treatment targeting both intracellular bacteria and oxidative stress. Supportive data demonstrated that combination treatment with antibiotic and NOX2 inhibitor significantly improved the survival rate of influenza and MRSA co-infected mice compared with antibiotic treatment alone. Specific aim 3 is to optimize this combination therapeutic approach based on the findings from studies proposed in aim 1 &2. The ultimate goal of this project is to establish the treatment strategy to restore antimicrobial activity but to control inflammatory lung damage during influenza and MRSA co-infection. The results achieved from these proposed studies will provide not only pivotal but also directly applicable information for the development of novel therapeutics to reduce the high mortality in patients.